Monitoring of cells

ABSTRACT

A method for monitoring cells in a microfluidic-device, wherein the device includes a chamber comprising a sensor, and the monitoring is under conditions such that attachment of cells to the surface of the chamber is inhibited.

FIELD OF THE INVENTION

This invention relates to a method for observing or monitoring cells, inparticular their metabolism, to optimising cell culture conditions, andfor high throughput screening, and to a device suitable for use in sucha method.

BACKGROUND OF THE INVENTION

A microfluidic device typically comprises at least one channel orchamber having a size that is equal to or less than 1 mm in at least onedimension. Of particular interest are microfluidic devices suitable forcarrying out and/or monitoring chemical and biological processes. Thesedevices can be used to simulate large-scale processes at a microscopiclevel, thus minimising volumes of fluids and reagents.

A microfluidic device may be used to monitor cell metabolism. Changes inthe external biological, chemical and physical milieu of a cellspecifies particular patterns of up- and down-regulated gene expressionand results in changes in the concentrations and fluxes of metaboliteswithin the cellular environment. All living cells take in nutrients andbreak them down to produce useful energy and waste products. Forexample, almost all cells change the pH of their environment by takingup alkaline or acidic nutrients or producing organic acids, CO₂ or NH₄+.Cell metabolism of this type can be monitored with a silicon-basedmicrophysiometer, which measures the rate at which eukaryotic cellschange the pH of their immediate environment in real time inthermoregulated microvolume flow chambers; see Wada et al, 1991, Clin.Chem. 37:600-601; and McConnell et al, 1992, Science 257:1906-1912.

This microphysiometer suffers from a number of limitations. For example,the light-addressable potentiometric sensor (LAPS), on which the systemis based, relies on expensive microengineered silicon technology and hasa relatively high microvolume (multiple μL). Furthermore, the devicerequires sophisticated instrumentation, uses biohostile inorganicsurfaces and, as currently configured, is able to monitor only a singleanalyte (pH).

Conventional cellular microfluidic devices comprise channels andchambers having a relatively large volume (of the order of μL). The sizeof a microfluidic device is preferably as small as possible, so thatreagent/sample quantities are minimised.

Since microfluidic technology deals with tiny volumes of reagents andfluids, the observation and monitoring of cells may depend critically onthe surface area/volume ratio of the microfluidic chambers and channels,and on the nature of the biofilm that forms on the inner surface of achannel or chamber.

Conventional microfluidic channels and chambers are typically restrictedto volumes of the order of μL. If the volume is smaller (say of theorder of nL), then the reduction in volume is not mirrored by areduction in surface area and the effect of uneven biofilm formation ismore pronounced.

The size of a microfluidic device may also be limited by the size of anysensor used. Other problems faced by microfluidic technology include theprovision of low-cost devices and microfluidic circuits, theestablishment of high-throughput methods for inoculation and thereal-time detection of cellular activity.

Conventional microfluidic devices are generally fabricated in glass,silica or silicon-based substrates using photolithographic processingtechniques to generate channels, reaction chambers or arrays. Whilstsuccessful prototype devices have been prepared using these substrates,the associated fabrication techniques, including mask production,channel etching and sealing, and bonding and packaging, are generallydifficult and/or expensive to implement.

The large-scale production of micromachined devices is hampered by thefact that wafer sizes are relatively small, the cost of processing isprohibitive, set-up, tooling, design changes and revisions are expensiveand the introduction of three dimensionality into the design requiresexpensive registration and etching or cutting facilities.

SUMMARY OF THE INVENTION

According to one aspect of the invention, cells are monitored in amicrofluidic device comprising a sensor. This is done under conditionssuch that attachment of cells to a wall of the device is inhibited.

According to another aspect of the invention, a microfluidic devicecomprises a chamber including a sensor and inlets for a sample and for agrowth medium. A wall of the device is such that, in use, attachment ofcells to the wall is inhibited.

The chamber is preferably in connection with a second, downstreamchamber. The second chamber may be used to detect cellular componentssuch as expressed proteins or enzymes. In this case, a device of theinvention may be considered to comprise a biotic (first) chamber inconnection with an abiotic (second) chamber.

By inhibiting attachment of the cell to a wall of the chamber, biofilmformation is minimised or inhibited. The size of the microfluidicchamber can be less critical than in conventional microfluidic devices,despite having a high surface area/volume ratio, because the effects ofbiofilm formation are markedly reduced even at small volumes (e.g. ofthe order of nL).

A device of the invention preferably comprises a plurality ofmicrofluidic chambers, which may be connected by microfluidic channels.Such an array may be used to study multi-factorial effectors such ascarbon/nitrogen sources, vitamin and mineral supplements and theireffect on microbial physiology.

A device of the invention may provide a rapid low-cost method ofinvestigating the performance of whole cell systems and may be utilisedin areas such as pharmaceuticals and biotechnology (whole cellscreening, drug discovery, genomics, proteomics and metabolomics, tissueculture, biotransformations, chiral drug production), food and beverage(growth conditions, media optimisation, fermented products such as aminoacids, vitamins, gums, acids), environmental (bioremediation) andacademia (microbiology, genomics, biochemistry).

The invention may allow significant cost and time reductions in reagentsand allow the optimisation of conditions for the growth of inter aliabacteria, fungi and mammalian cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A device of the invention comprises a microfluidic chamber including asensor and inlets for a sample and a growth medium, and preferably anoutlet. The reagents are preferably fed through one or more microfluidicchannels into the chamber. An outlet of the chamber may then lead,preferably via a microfluidic channel, to a waste reservoir or anotherchamber. An output channel may be used as an overflow and sampling lineand may be obstructed by a printed interdigitated pattern of closelyspaced islands to act as a filter to prevent egress of the growing cellsfrom the chamber.

A device of the invention is preferably formed using a plasticsmaterial. Plastics represent attractive materials for microfluidicsystems since they are less expensive and more easily manipulated thansilica/silicon-based substrates. A wide range of low-cost polymermaterials enables selection of the appropriate plastics for thermal andchemical resistance, photolithographic and silk screen printing, surfacederivation and multi-layer bonding. Plastics materials may include bothlinear and cross-linked, opaque and translucent plastics offering thepossibility of inert surfaces for non-aqueous media,chemically-modifiable surfaces and devices which can be interrogatedoptically; or which may be electrically conducting. Compared to currentglass, silica or silicon-based substrates, the use of plastics offersseveral distinct advantages including a feature resolution equivalent tosilicon-based technologies, short design cycles, low tooling costs andsubstantial design flexibility, and the possibility of fabricating manythousands of devices on a single plastic sheet. Electrical connectionscan be made directly to the device itself, without the requirement forbonding, mounting or packaging technologies. Examples of suitableplastics materials include polyolefins such as polypropylene (PP),polycarbonate, PDMS, polyvinyl chloride (PVC), polystyrene,fluoropolymers and acrylates such as polymethyl metacrylate (PMMA).

Selection of appropriate materials should take into account the relevantcharacteristics of mechanical, chemical, electrical, acoustic, optical,gas permeability, surface morphology, sterilisability, biocompatibility,and, impotrantly, the ability to join, etch or print onto them. Thepotential effects of additives such as stabilisers, fillers,plasticisers and colourants, e.g. on cellular growth and viability,should also be taken into consideration.

A device of the invention may comprise a multi-layer construction. Amaterial of the type described above is particularly suitable as asubstrate.

Such a substrate may have microfluidic chambers/channels formed in it.Preferably, however, a different, more tractable material, such as anepoxy resin, is formed as a “matrix” layer on the substrate and themicrofluidic chambers/channels are formed in that. If necessary ordesired, the walls of these features may then be treated or coated, inorder to inhibit cell attachment. The device may then be completed byproviding a further layer of a substrate-type material, on top of themicrofluidic-defining layer.

Each layer may be formed and joined together by known techniques.Suitable formation techniques include printing and moulding; adhesive orlamination may be used to join layers. Similarly, the microfluidic maybe formed by known microengineering procedures, including ablation usingan excimer or other laser.

The substrate is preferably impermeable. In order to introducesamples/reagents etc, or to allow monitoring or the sensor, one or moreholes may be punched or drilled in it, e.g. using a laser. Alternativelyor in addition, the substrate material should allow a needle to bepunched through it, through which materials are delivered.

The or each chamber typically has a volume of at least 50 nL, e.g. up to10 μL but often no more than 500 nL, 1 μL or 2 μL. In a 500 μm thicksubstrate, holes of width 1.0, 1.5 and 2.0 mm would result in chambershaving a volume of approximately 400 nL, 900 nL and 1.6 μL,respectively.

A feature of the invention is that attachment of cells to a wall of thechamber is inhibited. By inhibiting the attachment of cells on the wallsof the chamber, the formation of biofilm is controlled.

There are many methods of inhibiting cell attachment. These includecoating the inside of a chamber and/or channels with a hydrophilicmaterial, for example polyvinyl alcohol (PVA), and incorporating anon-metabolisable inhibitor of mannose-specific adhesin (e.g. methylα-D-mannopyranoside) in the growth medium.

The wall of the chamber is preferably smooth. A substrate such as PMMAor PP is smooth, and the smoothness of an epoxy resin can be controlled,e.g. by sonication, degassing, or centrifugation before application.Further, treatment with acid or alkali can help to minimise nucleationpoints.

Bacterial growth may be promoted by increasing the supply of oxygen tothe bioreactor chamber. The device preferably comprises an internalsurface layer of a gas-permeable material, e.g. providing permeabilityto oxygen, CO₂ or NH₃. Alternatively, the internal surface may beoxygen-impermeable, for an (obligate) anaerobe. Examples ofgas-permeable plastics include polyalkenes such as low-densitypolyethylene (LDPE) and polymethylpentene (PMP). These are alsooptically clear and chemically resistant. Polyalkenes are hydrophobic,“low-energy” plastics. For adhesion to an epoxy layer, some surfaceactivation is necessary. It is also desirable to make the surface ashydrophilic as possible to prevent bacterial adhesion during use. Thiscan be achieved by etching a polyalkene surface using an oxygen-plasmamethod; in this case, the polyalkene is preferably coated, e.g. withPVA, to prevent reversion to a hydrophobic surface. Alternatively, thesurface of the polyalkene can be treated with chromic acid; thisoxidises the surface of the plastic, rendering hydrophilicity. Thesetechniques have been shown to reduce the adhesion of bacteria such as E.coli and L. casei by up to 90%.

A device of the invention preferably comprises a fluoropolymer layer.Fluoropolymers such as fluorinated ethylene-polypropylene (FEP) haveexcellent gas-permeability and optical properties. However,fluoropolymers have a low reactivity and thus require pre-treatmentbefore application. One method is to use a reducing agent such as asodium napthalide complex. This agent is available in a range ofcommercial mixes (e.g. Tetra-Etch produced by W. L. Gore Associates).Controlled reoxidation, e.g. using an oxidising agent such as nitricacid may be required.

The comprehensive investigation of biological processes demands anappreciation of both the biotic and abiotic phases. The biotic phasedefines the cellular growth, metabolic and productivity parameters of abiological system, whilst the abiotic phase is defined by the productsof metabolism which may be chemical and/or physical.

The device preferably comprises a biotic chamber in combination with anabiotic chamber. The abiotic chamber may comprise capture, separationand analytical systems designed to target individual profiles ofproducts of the cellular process occurring in the biotic chamber. Inthis case, a bioreactor chamber may be viewed as a “quasi-chemostat”,wherein the liquid overflow provides a sample source for downstreamanalysis of the products of metabolism. Patterns of metabolic responseto nutritional and environmental stimuli may be investigated; theseobservations can then be used to make qualitative and quantitativejudgements about the microorganism under investigation. The results maythen be used to define the critical nutritional, environmental andtemporal components.

Similarly, a device of the invention may also be used to capturespecific proteins via appropriate affinity adsorbents or His-tag bindingagents and monitoring their presence with a suitable sensor, for examplea holographic or an acoustic sensor. The approach of using pulsedacoustic waves induced by magnetic direct generation is compatible withplastic substrates, since acoustic losses are considerably less thanthose experienced in the resonance format, and interrogation with acontinuous spectrum of frequencies is possible, thereby allowing“fingerprinting” of target proteins, cells or oligonucleotides.

The design of the device may be tailored to its intended application.This may involve the provision of multi-channel feed inputs for dosingof more than one growth medium such as different carbon and nitrogensources, vitamins, growth factors, mineral supplements and precursors,and additional sensors in the chamber. The chamber preferably comprisesa stirrer, preferably a magnetic bead 20-100 μm in diameter. Otherconsiderations include the printing and/or photolithographic etching ofthe microchamber, microchannels of high aspect ratio,microfluidic-driving mechanisms, flow characteristics, pumps, stirrersand retaining filters and the fabrication of downstream and analyticalmodules.

The chamber comprises one or more sensors for monitoring cellularactivity, for example by detecting changes in biomass (cell numbers) ortemperature and alterations in key attributes associated with growth(pH, dissolved oxygen, redox potential, substrate concentrations). Aplurality of sensors may be located adjacent to each other in a controlbinnacle in the bioreactor chamber. The sensor(s) may be placed aroundthe periphery of the well, thereby not only allowing a clear opticalpath length but also, in addition, acting as an actuator for a stirrer.A chamber may be fitted with a microscope (preferably equipped with adigital camera and imaging software) which may be used for monitoringeukaryotic cellular processes.

Holographic sensor technology is ideally suited to the presentinvention. A holographic sensor may be used to monitor a wide range ofanalytes, such as gases, ions, metabolites, antigens and whole cells, inreal-time, with rapid response times. The sensor may be sensitive tocombinations of analytes such as gases (e.g. O₂. CO₂), glucose, pH,lactate, glutamate/glutamine, temperature, redox, ions (e.g. ammoniumions) and cellular products (antibiotics, enzymes, expressed proteins).Suitable holograms of this type are described in WO-A-95/26499 andWO-A-99/63408.

Sensor electrodes can be used for the in situ measurement of pH,dissolved oxygen, glucose or other biochemical substrates. A pH-sensingelectrode may comprise metal oxides such as tantalum pentoxide (printedfrom tantalum(V) isopropoxide, sol-gel formation and curing at 100-110°C.) or the dioxides of iridium, platinum or ruthenium (printed frompre-sintered ruthenium dioxide hydrate mixed with a polymer paste in theratio 1:2 by weight). A dissolved oxygen-sensing electrode may comprisea gold or three-layer construction comprising a substrate plastic,platinum microarray electrode and silver/silver bromidepseudo-reference, and a gas-permeable PTFE membrane enclosing aninternal electrolyte gel. A temperature or redox potential may also beformed from printed platinum. Biomass may be measured by opticalinterrogation at 580-600 nm using an amber LED (y_(max)592 nm) as alight source, a silicon photodiode/amplifier as detector and fibre opticlight guides to convey the light into and out of the chamber. Furthertechniques that may be used include membrane fluorescence, colour changeetc.

A ruthenium-based sensor may also be used, in particular a fluorescencequenching ruthenium-based oxygen sensor. A range of such compounds witha ruthenium ion and three phenanthroline-based ligands is available andcan be immobilised in a polymer matrix for the purpose of determiningoxygen content. Other fluorescence-based sensors can be used; there is arange of compounds giving increases or decreases in fluorescence withchanges in ion concentrations, gas concentrations, pH etc. Similarlythere are a range of simple colour change reactions that can beharnessed, especially for variables such as pH.

Data from the chamber can be stored on a datalogger, preferablycomprising a computer installed with a suitable analogue interface. Theinterface circuitry is preferably flexible allowing simple high-quality,low-frequency measurements (e.g. every 15 s over a 48 hour period) orfor simultaneous “snapshots” of the signals (e.g. of over 400 sensors).The data produced by these circuits will be available to the computersuch that datalogging, analysis and real-time feedback control systems.The efficient investigation and exploitation of biotic and abioticprocesses using massively parallel cell culture systems requires theapplication of high-dimensionality data generation and analysis.Multi-factorial and other statistical methods are preferablyincorporated for experimental design, control and analysis. Multivariatemethods such as Principal Components Analysis can be used to deriveinformation and knowledge from the holographic and other analyticalresponse variable data sets. Algorithmic sensors and inferential enginesmay also be constructed combining disparate biotic and abiotic datastreams to provide information and knowledge on metabolic, physiologicaland cellular status.

A device of the invention may be in the form of an array of microfluidicchambers, optionally connected by microchannels. The array may be anarray of biotic and abiotic chambers. For example, such as array may beused to implement feed and management and control systems for monitoringthe performance of up to a multiplicity of chambers, wherein eachchamber on the array is provided with different growth media. Wherenecessary, careful consideration must be given to the configuration ofthe array to ensure that the microfluidic contacts and the scale-upissues associated with the macroscopic multiplexing of fluidics, opticalinterrogation systems and input/output contacts are achievable withinthe device. The array may be configured in a two-dimensional array,tape, disc or drum format, with any biotic, abiotic and analyticalphases arranged appropriately. More generally, the sensors may be in theform of an array within one or more chambers of which any pair may beinterconnected by a suitable channel. The layout of the array, e.g. inrows and columns, should merely be compatible with appropriate opticalor other detection apparatus.

The present invention is particularly relevant to the study of cellculture, e.g. tissue culture and fermentation processes. Currenttechnology for the study of fermentation development struggles to allowthe simultaneous study of a large number of metabolic effectors, eithersingly or in combinations of interacting factors. This is necessary inorder to define as rapidly as possible the fermentation protocol formaximum productivity. There is thus a need to devise a system that cansimultaneously test hundreds or thousands of effectors of microbialphysiology. This may be realised by using a device, preferably in theform of an array, of the invention.

The present invention may also be used in the study of filamentousmicroorganisms, biotransformation of precursor molecules, multipleculture inoculation (including spores), cell-cell signalling novelbioactive antibacterial compounds and metabolically engineered cells.Other applications of the present invention include the rapid analysisof mutationally segregate cells (in support of functional genomics),high-throughput screening of antibiotic production and susceptibility,automation of cellular bioassays for evaluating agonists/antagonists,cellular systems for the production of key biopharmaceutical compounds,measurement of cellular energy fluxes, metabolic and physiologicalstatus, mixed culture systems and their interactions, environmentalimpact of cytotoxic agents, receptor-mediated responses, subtypes andsignal transduction pathways and ligand-gated ion channels.

The following Examples illustrate the invention.

EXAMPLE 1

Microfluidic devices were produced using a polycarbonate substrateprinted with an epoxy resin. The resin layer was used to define inputand output microchannels and the microfluidic chamber. Hydrochloric acidwas introduced into the chamber of each device at a range of differentconcentrations (0.02 to 0.2M), and allowed to stand for 45 minutes tocure the epoxy groups. The devices were then rinsed with steriledistilled water until the pH of the rinse became the same as thebackground level of the water, and dried. A sample of E. coli cells anda growth medium was then introduced into the chamber and cultured.

Curing of the epoxy groups was found to significantly reduce theattachment of cells to the surface of the epoxy resin in the chamber,with little biofilm observed after 1 hour.

EXAMPLE 2

E. coli cells were cultured in non-agitated LB growth medium in threepolycarbonate/epoxy resin microfluidic devices. One device comprised LBcontaining additional mannose whilst in another device the LB comprisedthe non-metabolisable mannose analogue, methyl α-mannopyranoside. After4 hours incubation at 37° C., each device was stained with crystalviolet and the number of bacteria attached per field of vision (at 600×magnification) counted.

FIG. 1 shows that the presence of mannose and the mannose analogueresulted in a significant reduction in the level of bacterial adherence.

EXAMPLE 3

A microfluidic device was formed by laminating a polycarbonate substrateand epoxy and LDPE layers. The LDPE provided means for introduction of aneedle for material delivery. The formed epoxy was treated with acid andcoating with polyvinyl alcohol (PVA), which was subsequentlycross-linked with acidic glutaraldehyde. Excess glutaraldehyde was thenwashed out. Static E. coli/LB cultures were used to evaluate the degreeof biofilm formation.

FIG. 2 shows that the presence of the hydrophilic, cross-linked PVAsignificantly limits the extent of biofilm formation. For each pair ofcolumns (a, b and c), the left column shows the bacterial count on theepoxy whilst the right column shows the count on the polycarbonate.

EXAMPLE 4

A range of polycarbonate-backed, epoxy-coated devices was prepared.Two-part epoxy was prepared using a standard mixing method and aliquotswere sonicated to remove a portion of the air bubbles. After coatingeach device with PVA, the devices were separated according to the numberof ruptured air bubbles present on the surface. Sonication was found toreduce the number of ruptured air bubbles by approximately 50%. Thedevices were then used to ferment static terrific broth cultures of E.coli.

Close investigation of the ruptured air bubbles revealed that whilst themajority of bubbles did not show any increase in the level of attachedbacteria, some were covered by thick colonies of cells. These coveredbubbles displayed distinctive “sharp” characteristics, with the edgesdistinct and pronounced. Approximately 5% of ruptured bubbles fell intothis category.

FIG. 3 shows the number of bacteria attached to a polycarbonate/epoxyresin device having a “sharp” surface (produced by lamination with LDPE,which was removed once the epoxy was cured) and one having a “smooth”surface.

The smooth surface is less susceptible to bacterial adhesion and thusbiofilm formation.

EXAMPLE 5

Six polycarbonate-based microfluidic devices were produced comprising apolyalkene (LDPE) top coat. Two of the devices were treated with oxygenplasma for 30 seconds and another two for 90 seconds, to provide twosets of devices each treated to different extents. One set was thentreated with PVA;

-   -   the devices of the other set were not so treated. E. coli was        then cultured in the chamber of each device.

Polyalkenes are hydrophobic and thus encourage bacterial adhesion. Bycoating the polyalkene with hydrophilic PVA, the bacterial count wasexpected to be reduced.

FIG. 4 shows the initial bacteria count and that after 6 and 24 hours.

Both plasma-treatment of the polyalkene and adding PVA reduce the levelof bacterial adhesion.

EXAMPLE 6

Three polycarbonate/epoxy resin microfluidic devices were producedcomprising a fluorinated ethylene polypropylene (FEP) upper layer. Oneof the devices was formed by pre-treating the FEP with sodiumnapthalide. Another of the devices was formed by pre-treating FEP withthe naphthalene complex and coating with PVA. E. coli was then culturedin the microfluidic chamber of each device. Sodium naphthalene complexwas used to increase the reactivity of FEP and thus aid production ofthe device.

FIG. 5 shows the bacterial count of each device. Whilst coating the FEPlayer with sodium naphthalene complex does increase the number ofadhered bacteria, the total number of adhered cells is still in therange found for other plastics materials typically used in a device ofthe invention. Coating the FEP layer with PVA caused a valuablereduction in bacterial adhesion. FEP is a preferred material due to itsdesirable gas-permeability and optical properties.

EXAMPLE 7

Two polycarbonate microfluidic devices were produced comprisingoxygen-permeable top layers; one device comprised a polymethylpentene(TPX) top layer, the other an FEP top layer. E. coli was subsequentlycultured in the microfluidic chamber of each device.

It was found that the rapid aerobic growth phase of the facultativeanaerobe E. coli could be extended by using a more oxygen-permeableplastic.

TPX is more permeable to oxygen than FEP and, as FIG. 6 shows, supportsa longer aerobic exponential growth phase before oxygen becomes alimiting nutrient.

1. A method for monitoring cells in a microfluidic device, wherein thedevice includes a chamber comprising a sensor, and the monitoring isunder conditions such that attachment of cells to the surface of thechamber is inhibited.
 2. The method according to claim 1, wherein thechamber surface comprises a gas-permeable material.
 3. The methodaccording to claim 2, wherein the gas is selected from the groupconsisting of CO₂, NH₃, and O₂.
 4. The method according to claim 2,wherein the material is a fluropolymer.
 5. The method according to claim1, wherein the chamber surface comprises a hydrophilic material.
 6. Themethod according to claim 5, wherein the hydrophilic material ispolyvinyl alcohol.
 7. The method according to claim 1, wherein thechamber is formed in an epoxy resin coated on a plastics substrate. 8.The method according to claim 7, wherein the plastics substrate ispolycarbonate.
 9. The method according to claim 1, wherein the chambercomprises a plurality of sensors.
 10. The method according to claim 1,wherein the sensor is sensitive to oxygen, carbon dioxide, ammonium ionor pH.
 11. The method according to claim 1, wherein the sensor is anoptical sensor.
 12. The method according to claim 11, wherein the sensoris a holographic sensor.
 13. The method according to claim 1, whereinthe sensor is an electrochemical or acoustic sensor.
 14. The methodaccording to claim 1, wherein the sensor is sensitive to a reactant orproduct of fermentation.
 15. The method according to claim 1, whereinthe volume of the chamber is from 50 nL to 10 μL.
 16. The methodaccording to claim 1, which further comprises introducing growth mediuminto the chamber, wherein the sensor is sensitive to a reactant orproduct of cell growth.
 17. The method according to claim 16, whereinthe growth medium comprises a non-metabolisable mannose analogue. 18.The method according to claim 17, wherein the analogue is methylα-D-mannopyranoside.
 19. The method according to claim 1, which furthercomprises introducing a component of or derived from the cells into asecond microfluidic chamber comprising a sensor and in connection withthe first chamber detecting said component.
 20. The method according toclaim 19, wherein the component is a product of cell growth.
 21. Themethod according to claim 19, wherein the component is an expressedprotein or enzyme.
 22. The method according to claim 19, wherein thesensor of the second chamber is as defined in any of claims 10-15.
 23. Amicrofluidic device which comprises a chamber including a sensor andinlets for a sample and for a growth medium, wherein the chamber surfaceis such that, in use, attachment of cells thereto is inhibited.
 24. Thedevice according to claim 23, wherein the chamber surface comprises agas-permeable material.
 25. The device according to claim 23, whichcomprises a plurality of the chambers.
 26. The device according to claim25, wherein the chambers are in the form of an array.
 27. The deviceaccording to claim 25, wherein a pair of chambers is connected by achannel.
 28. The device, according to claim 23, wherein the material isa fluropolymer.
 29. The device, according to claim 23, wherein thechamber surface comprises a hydrophilic material.
 30. The device,according to claim 23, wherein the chamber is formed in an epoxy resincoated on a plastic substrate.
 31. The device, according to claim 23,wherein the sensor is sensitive to oxygen, carbon dioxide, ammonium ionor pH.
 32. The device, according to claim 23, wherein the sensor is anoptical sensor.
 33. The device, according to claim 23, wherein thesensor is a holographic sensor.
 34. The device, according to claim 23,wherein the sensor is an electrochemical or acoustic sensor.
 35. Thedevice, according to claim 23, wherein the sensor is sensitive to areactant or product of fermentation.